Mechanisms of calcium handling during a malignant hyperthermia event.

Abstract

Malignant hyperthermia (MH) is a clinical syndrome of the skeletal muscle that presents as a hypermetabolic response to volatile gas anaesthetics such as halothane, isoflurane, and to the depolarizing muscle relaxant succinylcholine. Genetic predisposition mainly arises from mutations in the gene for skeletal muscle ryanodine receptor (RyR1); a calcium channel that is tightly regulated to release Ca 2+ for muscle contraction. MH susceptible RyR1 are more prone to open by RyR1 agonist, resulting in the uncontrolled release of Ca 2+ from the sarcoplasmic reticulum (SR). The hypermetabolic state is due to the excessive consumption of adenosine triphosphate (ATP) as the muscle works to clear the high cytosolic Ca 2+. Dantrolene has been used to great effect by clinicians in treating MH. However the mechanism of its action remains highly contested. This thesis examines Ca 2+ handling in muscle fibers where RyR1 activity is changed due to mutations. RyR1 activity was also acutely changed by altering the cytoplasmic environment and by treating the muscle fiber with varying RyR1 modulators. The release of SR Ca 2+ was monitored using fluorescent Ca 2+ indicators in mechanically skinned fibers isolated from rat, toad and human muscle susceptible to MH. The results show that the inhibitory effect of dantrolene is dependent on cytosolic concentrations of Mg 2+ ([Mg 2+ ]cyto), an endogenous negative regulator of the RyR1. Furthermore, it was demonstrated in human muscle susceptible to MH that dantrolene was ineffective at reducing halothane-induced Ca 2+ release in the presence of resting levels of [Mg 2+ ]cyto (1 mM). These results suggest that raised [Mg 2+ ]cyto, a condition which is likely to arise during an MH event, works synergistically with dantrolene to inhibit Ca 2+ release from the RyR1. It was also demonstrated that halothane induced Ca 2+ release took the form of a repetitive Ca 2+ wave in human MH, and toad muscle. A close examination of the release mechanism in human MH muscle revealed that the release of SR Ca 2+ and the mechanism underlying the propagation of the Ca 2+ wave was not due to Ca 2+-induced-Ca 2+-release (CICR). These results were contrasted to the observation made in toad skeletal muscle which showed the involvement of CICR. However, it was found that both luminal SR and cytosolic Ca 2+ plays an important role in the mechanisms involved in overactive Ca 2+ release. The demonstration of the Mg 2+ dependence on dantrolene inhibition of the RyR1 emphasized the importance of studying the RyR1 under near physiological conditions. Utilizing a novel approach by trapping Ca 2+ indicator in either the SR or the transverse tubule (t-system) of mechanically skinned rat skeletal muscle fibers, it was determined if Publications during candidature Choi RH, Koenig X & Launikonis BS. (2017). Dantrolene requires Mg 2+ to arrest malignant hyperthermia. Proceedings of the National Academy of Sciences 114, 4811-4815. Conference abstracts R.H. Choi, and B.S. Launikonis (2017). Dantrolene requires Mg 2+ to arrest mal...